Antenatal creatine supplementation reduces persistent fetal lung inflammation and oxidative stress in an ovine model of chorioamnionitis.

Background Chorioamnionitis is a common antecedent of preterm birth and induces inflammation and oxidative stress in the fetal lungs. Reducing inflammation and oxidative stress in the fetal lungs may improve respiratory outcomes in preterm infants. Creatine is an organic acid with known anti-inflammatory and antioxidant properties. Objective To evaluate the efficacy of direct fetal creatine supplementation to reduce inflammation and oxidative stress in fetal lungs arising from an in utero pro-inflammatory stimulus. Methods Fetal lambs (n=51) were instrumented at 90 days gestation to receive a continuous infusion of creatine monohydrate (6 mgkg-1h-1) or saline for 17 days. Maternal chorioamnionitis was induced with intra-amniotic lipopolysaccharide (LPS; 1 mg, O55:H6) or saline seven days before delivery at 110 days gestation. Tissue creatine content was assessed with capillary electrophoresis, and inflammatory markers were analyzed with Luminex Magpix and immunohistochemistry. Oxidative stress was measured as the level of protein thiol oxidation. The effects of LPS and creatine were analyzed using a 2-way ANOVA. Results Fetal creatine supplementation increased lung creatine content by 149% (PCr<0.0001) and had no adverse effects on lung morphology. LPS-exposed groups showed increased levels of interleukin-8 in the bronchoalveolar lavage (PLPS<0.0001) and increased levels of CD45+ leukocytes (PLPS<0.0001) and MPO+ (PLPS<0.0001) cells in the lung parenchyma. Creatine supplementation significantly reduced the levels of CD45+ (PCr=0.045) and MPO+ cells (PCr=0.012) in the lungs and reduced thiol oxidation in plasma (PCr<0.01) and lung tissue (PCr=0.02). Conclusion Fetal creatine supplementation reduced markers of inflammation and oxidative stress in the fetal lungs arising from chorioamnionitis.

Biological Variation in Biochemistry Analytes in Laboratory Guinea Pigs (Cavia porcellus).

Biological variation (BV) describes the physiological random fluctuation around a homeostatic set point, which is a characteristic of all blood measurands (analytes). That variation may impact the clinical relevance of the changes that are observed in the serial results for an individual. Biological variation is represented mathematically by the coefficient of variation (CV) and occurs within each individual (CVI) and between individuals in a population (CVG). Biological variation data can be used to assess whether population-based reference or subject-based reference intervals should be used for the interpretation of laboratory results through the calculation of the index of individuality (IoI). This study aimed to determine the biological variations, calculate the IoI and reference change values (RCV) of clinical chemistry analytes in an outbred strain colony of Hartley guinea pigs (GPs), and set the quality specifications for clinical chemistry analytes. Blood was collected from 16 healthy adult laboratory colony GPs via jugular venipuncture at weekly intervals over six weeks. All the samples were frozen and analyzed in a single run. Analytical, CVI, and CVG biological variations, together with the IoI and RCV, were calculated for each measurand. Based on the estimated BV, the calculated IoI was low for glucose, so individual reference intervals (RCV) should be used. The majority of the measurands should be interpreted using both population-based and subject-based reference intervals as the IoIs were intermediate.

Injury to the tunica media initiates atherogenesis in the presence of hyperlipidemia.

Background and aims: Fatty streaks initiating the formation of atheromatous plaque appear in the tunica intima. The tunica media is not known to be a nidus for lipid accumulation initiating atherogenesis. We assessed changes to the tunica media in response to a micro-injury produced in the pig aorta. In addition, we assessed human carotid endarterectomy plaques for indication of atheroma initiation in the tunica media. Methods: Three healthy landrace female pigs underwent laparotomy to inject autologous blood and create micro-hematomas at 6 sites within the tunica media of the infrarenal abdominal aorta. These pigs were fed a high-fat diet (HFD) for 4-12 weeks. Post-mortem aortas from all pigs, including a control group of healthy pigs, were serially stained to detect lipid deposits, vasa vasora (VV), immune cell infiltration and inflammatory markers, as well as changes to the vascular smooth muscle cell (vSMC) compartment. Moreover, 25 human carotid endarterectomy (CEA) specimens were evaluated for their lipid composition in the tunica media and intima. Results: High lipid clusters, VV density, and immune cell infiltrates were consistently observed at 5 out of 6 injection sites under prolonged hyperlipidemia. The hyperlipidemic diet also affected the vSMC compartment in the tunica media adjacent to the tunica adventitia, which correlated with VV invasion and immune cell infiltration. Analysis of human carotid specimens post-CEA indicated that 32% of patients had significantly greater atheroma in the tunica media than in the arterial intima. Conclusion: The arterial intima is not the only site for atherosclerosis initiation. We show that injury to the media can trigger atherogenesis.

Continuous Telemetric In Utero Tracheal Pressure Measurements in Fetal Lambs.

Normal in utero lung development and growth rely upon the expansion of airspaces and the controlled efflux of lung liquid into the amniotic space. Infants with congenital diaphragmatic hernia (CDH) also have lung hypoplasia due to occupation of the chest cavity by the stomach and bowel and, in the most severe cases, the liver. Balloon tracheal occlusion reduces the severity of lung hypoplasia in fetuses with CDH but increases the risk of premature birth. Understanding the optimal occlusion pressure and duration required to improve lung hypoplasia with tracheal occlusion is essential to improving in utero corrective treatments for CDH. The study reports a new method for continuous measurement of the intratracheal and amniotic pressures in an unoccluded and occluded fetal lamb surgical model of CDH. Time-pregnant Merino ewes underwent two recovery hysterotomies: the first at ~80 days of gestation to create the CDH, and the second at ~101 days of gestation to occlude the fetal trachea and implant an intratracheal and amniotic pressure measurement device. Lambs were delivered at ~142 days, and the pressure measurement device was removed and cleaned. The data were downloaded and filtered using a 6 h window. Transrespiratory pressure was calculated.

Investigation of a method for long-term preservation of the vascular allograft.

BACKGROUND/OBJECTIVE: During multiple organ procurement, blood vessels are routinely retrieved and stored in University of Wisconsin solution and then discarded after two weeks, if not used at organ transplantation owing to lack of a method for long-term preservation. Therefore, the aim of this study is to investigate a method for long-term preservation of vascular allografts in ethanol. METHODS: Aorta and vena cava allografts were retrieved and stored in 75% ethanol solution for 12 months at 4°C. Four pigs were divided into two groups. A segment of aorta was excised and replaced by insertion of preserved aorta graft (Group A) or vena cava graft (Group V). The pigs were observed for six weeks. A laparotomy was performed and the vascular graft was harvested for histopathology followed by euthanasia at the end of study. RESULTS: Three pigs recovered uneventfully, while one pig died from venous graft rupture in the third week after surgery. There was no aneurysmal formation or thrombosis in the grafts. Some calcification was seen over aorta allograft. On histopathology, the elastic pattern was almost normal, although the endothelial cells degenerated after preservation. After implantation, the formation of the endothelium cell-like layer was seen in both aorta and vena cava allografts. CONCLUSION: Vascular allografts were functional after preservation for 12 months. The vena cava grafts had much less wall calcification than the aorta grafts. Further studies are necessary to investigate vascular graft remodelling with a longer observation period after implantation.

Oxygen delivery by mask improves the PaO2 of pregnant ewes during short term anaesthesia for caesarean delivery of preterm lambs.

The aim of this study was to determine if oxygen supplementation improved the PaO2 of pregnant ewes during short anaesthesia, when compared to a previous study where oxygen was not provided (Musk and Kemp, 2018). Twenty-six pregnant Merino cross ewes at 121-123 days of gestation were anaesthetised with intravenous midazolam and ketamine for subarachnoid administration of 60 mg of lignocaine and caesarean delivery of the preterm lamb. 100% oxygen was administered to the ewe by a face mask. Arterial blood samples were collected from the ewe immediately after delivery of the foetus. The ewes weighed 60.7 ± 5.5 kg and received 0.51 (0.47-0.58) mg/kg of midazolam and 10.3 (9.4-11.6) mg/kg of ketamine intravenously. The PaO2 of ewes receiving oxygen by face mask was higher than previously reported [92.6 ± 44.0 mmHg compared to 45.2 ± 11.8 mmHg (Musk and Kemp, 2018) (p = 0.0007)]. Oxygen delivery by mask improved the PaO2 of pregnant ewes during short term anaesthesia.

Maternal-fetal Blood Major Crossmatching in Merino Sheep.

To determine the incidence of ex vivo incompatibility between ovine maternal RBCs and fetal plasma, we performed cross-matching of blood samples from ewes and from lambs delivered by cesarean section. Twenty-one date-mated singleton pregnant Merino ewes were anesthetized for cesarean delivery of the fetus. At the time of delivery, paired maternal and fetal blood samples were collected and subsequently separated for storage as packed red blood cells and fresh frozen plasma. Gel column major cross matching was performed within 2 wk. All fetus-dam crossmatches were major crossmatches, combining fetal (recipient) plasma with dam (donor) RBCs. 172 individual dam-dam cross matches were performed. Two of these tests were incompatible (1.2%). In addition, 19 fetal blood samples collected immediately after cesarean delivery were crossmatched with 21 maternal samples to generate 174 maternal-fetal individual cross matches. No maternal-fetal incompatibility reactions were observed. The results of this study demonstrate that all maternal donors and fetal recipients were compatible. In addition, the incidence of an incompatible crossmatch between adult ewes was 1.2%. These data suggest that lambs may not be born with antibodies against other blood types, but rather may acquire such antibodies at some time during early life. In addition, these data suggest the risk of incompatibility reactions between ewes of a similar breed and from a single farm of origin is very low.

Anaemia and Hypoproteinaemia in Pregnant Sheep during Anaesthesia.

The aim of this study was to document the packed cell volume (PCV), haemoglobin concentration and total protein concentration of maternal blood before, during and after anaesthesia. Six singleton Merino-cross pregnant ewes at 116-117 days of gestation were premedicated with intramuscular acepromazine (0.02 mg/kg) and buprenorphine (0.01 mg/kg), and anaesthesia was induced with intravenous midazolam and ketamine. Anaesthesia was maintained with isoflurane in 100% oxygen. Serial blood samples were collected the day before anaesthesia (baseline), immediately prior to induction of anaesthesia (pre-op), at the end of the procedure (intra-op) and the following day (post-op). There was a significant change in the PCV during the study (p = 0.003) with an initial decrease of 12.5% from the baseline (0.36 (0.36-0.4) to 0.315 (0.29-0.34), p = 0.044), a further intraoperative decrease of 41.7% from the baseline (0.21 (0.195-0.245), p = 0.002) and an increase the day afterwards (0.3 (0.285-0.35), p > 0.99 compared with baseline). The haemoglobin concentration also changed (p < 0.0001) (baseline: 114 (111.8-123); pre-op: 97 (77.25-104.5), 14.9% decrease, p = 0.022; intra-op: 70 (61.5-83.25), 38.5% decrease, p = 0.0009; post-op: 101.5 (96.25-114) g/L, p > 0.99). Likewise the change in total protein during the study was significant (p = 0.0003) and decreased from the baseline [70 (67.25-70.75) g/L] prior to anaesthesia (61 (58.25-64.5) g/L, 12.9% decrease, p = 0.0437) and further during anaesthesia (55.5 (53.75-63.25) g/L, 20.7% decrease, p = 0.0021) with an increase [63 (61.25-67) g/L, p > 0.99] on the first post-op day. In conclusion, intraoperative anaemia and hypoproteinaemia occurred in this study. These alterations are attributed to a combination of the side effects of acepromazine and haemodilution.

Multiple refinements to the anaesthetic protocol for laparoscopic orthotopic kidney auto-transplantation in a pig model.

Fifty-four pigs ( Sus scrofa, Large White cross) from a commercial pig farm were anaesthetised over a six-year period from October 2011. The procedural stages included initial instrumentation for intra-operative monitoring, laparoscopic nephrectomy, preparation of the kidney graft, orthotopic auto-transplantation by either a laparoscopic approach or an open surgical approach, and ligation of the contralateral ureter. During the evolution and establishment of this pig model multiple refinements were introduced: (1) a heat pad was changed to a circulating warm air blanket; (2) routine administration of anticholinesterase and antimuscarinic drugs for reversal of neuromuscular blockade; (3) a technique for urethral catheterisation was developed; (4) ultrasound guidance for placement of a central venous line was learned; (5) intravenous infusions of morphine and ketamine were instituted for a more stable and balanced anaesthetic protocol; and (6) post-operative monitoring was performed by two technical staff for at least the first six hours after extubation of the trachea. The combination of refinements to the anaesthesia protocol for laparoscopic orthotopic kidney auto-transplantation over the first 54 pigs has translated to improved outcomes. This conclusion is entirely subjective as accurate measurement of the impact of each refinement is impossible. Nevertheless, refinement is a continuous process and should be applied to the entire range of procedures involved in animal use for scientific purposes.

Play-Modified One-Session Treatment for Young Children with a Specific Phobia of Dogs: A Multiple Baseline Case Series.

The one-session treatment (OST) approach for SPs is deemed well-established, and has been found to be highly effective for older children and adults; however, has not yet been trialled with very young children. The present study examines the preliminary effectiveness of play-modified OST for young children with a SP of dogs, using a multiple baseline controlled case series design. Treatment involved play modified one-session of intensive cognitive-behavioural therapy (OST plus Play) which was followed by brief telephone delivered maintenance calls over the 3 weeks immediately following treatment. Four young children (4 years of age) participated and symptoms were assessed at pre-treatment, across a 1-3 week baseline phase, immediately following the OST plus Play, and at 1 and 3 months follow-up. Visual inspection provided evidence for stability of symptoms across the baseline phase, followed by reductions in symptoms over the course of treatment and follow-up. Non-parametric analyses offered further support, with significant improvements in following the intensive OST plus Play intervention.

Cognitive Behavioral Therapy in a One-Session Treatment for a Preschooler With Specific Phobias.

Anxiety disorders among preschool-aged children are as prevalent as in older children, yet younger children are even less likely to receive treatment for these frequently impairing childhood disorders (Egger & Angold, 2006). Specific phobias (SPs) are often the earliest form of anxiety to onset, affect 2.3% of preschoolers (Egger & Angold, 2006), and tend to be stable from 3 years of age to at least 6 years of age (Bufferd, Dougherty, Carlson, Rose, & Klein, 2012). Recently, our group developed and piloted a modified intensive one-session treatment (OST) incorporating play therapy for preschoolers with SP (Farrell, Kershaw, & Ollendick, in press). Given that highly fearful young children may find concentrated, rapid exposure therapy highly confronting, we integrated play therapy into the commencement of this treatment to (a) assist with rapid rapport building with the therapist and (b) enhance the child's motivation through engaging them with play. An initial, controlled, baseline case series (N = 4) provided preliminary evidence of the feasibility, acceptability, and effectiveness of this innovative one-session (3 hours) therapy-POP! Pre-schoolers Overcoming Phobias (POP; Farrell et al., in press). The approach combines child-centered, nondirective play therapy (during Hour 1) and empirically supported OST (during Hours 2 and 3), with 4-year-old preschool children with clinical SPs of dogs. This article presents a case description of POP, describing the assessment approach, cognitive-behavioral case formulation, and intensive treatment of a 4-year-old girl who presented with a severe SP of dogs, along with various other comorbid SPs.

Localization and detection of breast cancer tumors with digital image elasto-tomography.

Digital Image Elasto-Tomography (DIET) is a novel elastic contrast based breast imaging method using time-harmonic motion data obtained from a calibrated array of high resolution digital cameras scanning the tissue surface. The method is currently undergoing initial clinical testing and preliminary results in cases of malignant breast tumors are now available. The method has proved capable of detecting and localizing the stiff lesions within the heterogeneous tissue structure of the beast through the use of an evolution based optimization algorithm implemented in linear finite elements. The method has also proved successful at detecting both inclusion and non-inclusion cases in specially constructed tissue mimicking silicon phantoms.